Back to EveryPatent.com
United States Patent | 5,783,565 |
Lee ,   et al. | July 21, 1998 |
Novel cationic amphiphiles are provided that facilitate transport of biologically active (therapeutic) molecules into cells. The amphiphiles contain lipophilic groups derived from steroids, from mono or dialkylamines, or from alkyl or acyl groups; and cationic groups, protonatable at physiological pH, derived from amines, alkylamines or polyalkylamines. There are provided also therapeutic compositions prepared typically by contacting a dispersion of one or more cationic amphiphiles with the therapeutic molecules. Therapeutic molecules that can be delivered into cells according to the practice of the invention include DNA, RNA, and polypeptides. Representative uses of the therapeutic compositions of the invention include providing gene therapy, and delivery of antisense polynucleotides or biologically active polypeptides to cells. With respect to therapeutic compositions for gene therapy, the DNA is provided typically in the form of a plasmid for complexing with the cationic amphiphile. Novel and highly effective plasmid constructs are also disclosed, including those that are particularly effective at providing gene therapy for clinical conditions complicated by inflammation. Additionally, targeting of organs for gene therapy by intravenous administration of therapeutic compositions is described.
Inventors: | Lee; Edward R. (Quincy, MA); Harris; David J. (Lexington, MA); Siegel; Craig S. (Woburn, MA); Cheng; Seng H. (Wellesley, MA); Eastman; Simon J. (Marlboro, MA); Marshall; John (Milford, MA); Scheule; Ronald K. (Hopkinton, MA) |
Assignee: | Genzyme Corporation (Framingham, MA) |
Appl. No.: | 595375 |
Filed: | February 1, 1996 |
Current U.S. Class: | 514/44; 424/450; 516/DIG.7; 536/23.1; 552/544 |
Intern'l Class: | A61K 009/127; A61K 048/00; B01F 017/16; C12N 015/00 |
Field of Search: | 514/44 552/544 252/357 424/450 |
4394448 | Jul., 1983 | Szoka et al. | |
4544545 | Oct., 1985 | Ryan et al. | |
4897355 | Jan., 1990 | Eppstein et al. | |
4958013 | Sep., 1990 | Letsinger. | |
4971803 | Nov., 1990 | Li et al. | |
5004737 | Apr., 1991 | Kim et al. | |
5171678 | Dec., 1992 | Behr et al. | |
5264618 | Nov., 1993 | Felgner et al. | |
5283185 | Feb., 1994 | Epand et al. | |
5334761 | Aug., 1994 | Gebeyehu et al. | |
5416203 | May., 1995 | Letsinger. | |
5614503 | Mar., 1997 | Chaudhary et al. | 514/44. |
Foreign Patent Documents | |||
0 446 017 A1 | Sep., 1991 | EP. | |
0 490 379 A3 | Jun., 1992 | EP. | |
61-7292 | Jan., 1986 | JP. | |
WO 88/00824 | Feb., 1988 | WO. | |
WO 94/05624 | Mar., 1994 | WO. | |
WO 94/20520 | Sep., 1994 | WO. | |
WO 95/02698 | Jan., 1995 | WO. | |
WO 95/21812 | May., 1995 | WO. | |
WO 95/14651 | Jun., 1995 | WO. | |
WO 95/14381 | Jun., 1995 | WO. | |
WO 95/14380 | Jun., 1995 | WO. | |
WO 95/17378 | Jun., 1995 | WO. | |
WO 95/24222 | Sep., 1995 | WO. | |
WO 95/31557 | Nov., 1995 | WO. | |
WO 96/03977 | Feb., 1996 | WO. | |
WO 96/10038 | Apr., 1996 | WO. | |
WO 96/20208 | Jul., 1996 | WO. |
K. Patel et al., "Modification of Vesicle Surfaces with Amphiphilic Sterols. Effect on Permeability and In Vivo Tissue Distribution", Biochimica et Biophysica Acta, 814, 1985, pp. 256-264. K. Moore et al., "Squalamine: An Aminosterol Antibiotic from the Shark", Proceedings of the National Academy of Sciences USA, 90, 1993, pp. 1354-1358. J. Guy-Caffey et al., "Novel Polyaminolipids Enhance the Cellular Uptake of Oligonucleotides", The Journal of Biological Chemistry, 270, 1995, pp. 31391-31396. J. Janne, et al., "Polyamines: From Molecular Biology to Clinical Applications", Annals of Medicine, 23, 1991, pp. 241-259. N. Caplen, et al., "Liposome Mediated CFTR Gene Transfer to the Nasal Epithelium of Patients with Cystic Fibrosis", Nature Medicine, 1, 1995, pp. 39-46. J-S. Remy et al., "Gene Transfer with a Series of Lipophilic DNA Binding Molecules", Bioconjugate Chemistry, 5, 1994, pp. 647-654. J-P. Behr, "Gene Transfer with Synthetic Cationic Amphiphiles: Prospects for Gene Therapy", Bioconjugate Chemistry, 5, 1994, pp. 382-389. J. Felgner, et al., "Enhanced Gene Delivery and Mechanism Studies with a Novel Series of Cationic Lipid Formulations", The Journal of Biological Chemistry, 269, 1994, pp. 2550-2561. F. Barthel et al., "Gene Transfer Optimization with Lipospermine-Coated DNA", DNA and Cell Biology, 12, 1993, pp. 553-560. J-P. Behr, "Synthetic Gene-Transfer Vectors", Accounts of Chemical Research, 26, 1993, pp. 274-278. P. Hoet, et al., "Kinetics and Cellular Localization of Putrescine Uptake in Human Lung Tissue", Thorax, 48, 1993, pp. 1235-1241. J. Felgner, et al., "Cationic Lipid-Mediated Delivery of Polynucleotides", Methods (A Companion to Methods in Enzymology), 5, 1993, pp. 67-75. X. Gao, et al., "A Novel Cationic Liposome Reagent for Efficient Transfection of Mammalian Cells", Biochemical and Biophysical Research Communications, 179, 1991, pp. 280-285. J. Rose, et al., "A New Cationic Liposome Reagent Mediating Nearly Quantitative of Animal Cells", Biotechniques, 10, 1991, pp. 520-525. J. Loeffler, et al., "Lipopolyamine-Mediated Transfection Allows Gene Expression Studies in Primary Neuronal Cells", Journal of Neurochemistry, 54, 1990, pp. 1812-1815. J. Cheetham, et al., "Cholesterol Sulfate Inhibits the Fusion of Sendai Virus to Biological and Model Membranes", The Journal of Biological Chemistry, 265, 1990, pp. 12404-12409. J-P. Behr, et al., "Efficient Gene Transfer into Mammalian Primary Endocrine Cells with Lipopolyamine-Coated DNA", Proceedings of the National Academy of Sciences, USA, 86, 1989, pp. 6982-6986. R. Letsinger, et al., "Cholesteryl-Conjugated Oligonucleotides: Synthesis, Properties, and Activity as Inhibitors of Replication of Human Immunodeficiency Virus in Cell Culture", Proceedings of the National Academy of Sciences, USA, 86, 1989, pp. 6553-6556. L. Stamatos, et al., "Interactions of Cationic Lipid Vesicles with Negatively Charged Phospholipid Vesicles and Biological Membranes", Biochemistry, 27, 1988, pp. 3917-3925. P. Felgner, et al., "Lipofection: A Highly Efficient, Lipid-Mediated DNA-Transfection Procedure", Proceedings of the National Acadmeny of Sciences, USA, 84, 1987, pp. 7413-7417. R. Rando, et al., "The Synthesis and Properties of a Functional Fluorescent Cholesterol Analog", Biochemica et Biophysica Acta, 684, 1982, pp. 12-20. A. Pegg, "Polyamine Metabolism and its Importance in Neoplastic Growth and as a Target for Chemotherapy", Cancer Research, 48, 1988, pp. 759-774. R. Kameji, et al., "Spermidine Uptake by Type II Pulmonary Epithelial Cells in Primary Culture", American Journal of Physiology, 256, 1989, pp. C161-C167. Jayaraman, K. et al. Approaches to enhancing the binding affinity and nuclease stability of triplex forming oligonucleotides. Nucleosides and Nucleotides 14:951-955, Jun. 13, 1995. |
__________________________________________________________________________ For R.sup.1 and/or R.sup.2 (1) --NH-- (2) --NH--(CH.sub.2).sub.(2) -- (3) --NH--(CH.sub.2).sub.(3) -- (4) --NH--(CH.sub.2).sub.(4) -- (5) --NH--(CH.sub.2).sub.(6) -- (6) --NH--(CH.sub.2).sub.(3) --NH--(CH.sub.2).sub.(4) -- (7) --NH--(CH.sub.2).sub.(2) --NH--(CH.sub.2).sub.(2) -- (8) --NH--(CH.sub.2).sub.(4) --NH--(CH.sub.2).sub.(3) -- (9) --NH--(CH.sub.2).sub.(y) --NH--(CH.sub.2).sub.(z) -- (10) --NH--(CH.sub.2).sub.(x) --NH--(CH.sub.2).sub.(y) --NH--(CH.sub.2).sub. (z) -- (11) --NH--(CH.sub.2).sub.(w) --NH--(CH.sub.2).sub.(x) --NH--(CH.sub.2).sub. (y) --NH--(CH.sub.2).sub.(z) -- (12) --NH--(CH.sub.2).sub.(v) --NH--(CH.sub.2).sub.(w) --NH--(CH.sub.2).sub. (x) --NH--(CH.sub.2).sub.(y) --NH--(CH.sub.2).sub.(z) -- (13) --›NH--(CH.sub.2).sub.(w) !.sub.m --›NH--(CH.sub.2).sub.(x)!.sub.n --››CH.sub.3 (CH.sub.2).sub.y !N!--(CH.sub.2).sub.2).sub.z -- (14) --›NH--(CH.sub.2).sub.(x) !.sub.n --›CH.sub.3 (CH.sub.2).sub.y !N!--(CH.sub.2).sub.z -- (15) --›NH--(CH.sub.2).sub.(w) !.sub.m --›NH--(CH.sub.2).sub.(x) !.sub.n --››CH.sub.3 (CH.sub.2).sub.y !N!--(CH.sub.2).sub.z -- (16) --››CH.sub.3 (CH.sub.2).sub.x !›CH.sub.3 (CH.sub.2).sub.y !N!--(CH.sub. 2).sub.z -- (17) --NH--(CH.sub.2).sub.(z) --NH-- (18) --NH--(CH.sub.2).sub.(y) --NH--(CH.sub.2).sub.(z) --NH-- (19) --NH--(CH.sub.2).sub.(y) --CH.dbd.CH--(CH.sub.2).sub.z -- (20) --›NH--(CH.sub.2).sub.(w) !.sub.p --››CH.sub.3 (CH.sub.2).sub.x !N!--(CH.sub.2).sub.y --›NH--(CH.sub.2).sub.(z) !.sub.q -- For R.sup.3 and/or R.sup.4 (1) H-- (2) CH.sub.3 -- (3) CH.sub.3 --(CH.sub.2).sub.2 -- (4) CH.sub.3 --(CH.sub.2).sub.4 -- (5) CH.sub.3 --(CH.sub.2).sub.z -- (6) CH.sub.3 --›CH.sub.3 --(CH.sub.2).sub.z !CH-- (7) CH.sub.3 --›CH.sub.3 --(CH.sub.2).sub.2 !CH-- (8) CH.sub.3 --››CH.sub.3 --(CH.sub.2).sub.y !›CH.sub.3 --(CH.sub.2).sub.z !!C-- (9) CH.sub.3 --(CH.sub.2).sub.z --CH.dbd.CH--CH.sub.2 -- (10) CH.sub.3 --›CH.sub.3 --(CH.sub.2).sub.y --CH.dbd.CH--(CH.sub.2).sub.z !CH-- (11) CH.sub.3 --››CH.sub.3 --(CH.sub.2).sub.w --CH.dbd.CH--(CH.sub.2).sub.x !›CH.sub.3 --(CH.sub.2).sub.y --CH.dbd.CH--(CH.sub.2).sub.z !!CH-- (12) CH.sub.3 --›CH.sub.3 --(CH.sub.2).sub.y !CH--(CH.sub.2).sub.z -- __________________________________________________________________________
TABLE 2 ______________________________________ For "Z" ______________________________________ (1) CH.sub.3 --(CH.sub.2).sub.13 --NH-- (2) CH.sub.3 --(CH.sub.2).sub.z --NH-- (3) ››CH.sub.3 (CH.sub.2).sub.17 !›CH.sub.3 (CH.sub.2).sub.17 !!N-- (4) ››CH.sub.3 (CH.sub.2).sub.11 !›CH.sub.3 (CH.sub.2).sub.11 !!N-- (5) ››CH.sub.3 (CH.sub.2).sub.9 !›CH.sub.3 (CH.sub.2).sub.9 !!N-- (6) ››CH.sub.3 (CH.sub.2).sub.x !›CH.sub.3 (CH.sub.2).sub.y !!N-- (7) ››CH.sub.3 (CH.sub.2).sub.x !›CH.sub.3 (CH.sub.2).sub.y CH.dbd.CH(CH. sub.2).sub.z !!N-- (8) ››CH.sub.3 (CH.sub.2).sub.w !›CH.sub.3 (CH.sub.2).sub.x CH.dbd.CH(CH. sub.2).sub.y CH.dbd.CH(CH.sub.2).sub.z !!N-- ______________________________________